Molecules that bind CD180, compositions and methods of use

ABSTRACT

The present invention provides novel CD180 binding molecules, methods for their identification, and methods for their use.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This invention was made with U.S. government support under DE16381,AI44257, and AI85311 awarded by the National Institutes of Health. TheU.S. Government has certain rights in the invention.

RELATED APPLICATIONS

This application claims prior to U.S. Provisional Patent ApplicationSer. No. 61/307,801 filed Feb. 24, 2010, which is incorporated byreference herein in its entirety.

TECHNICAL FIELD

The present disclosure relates to regulation of activation and functionof antigen-presenting cells such as B cells, myeloid cells, anddendritic cells. The disclosure provides compositions that activateCD180 and methods for their use. The disclosure also provides a methodfor stimulation of polyclonal immunoglobulin production for potentialtherapeutic application by binding to the CD180 receptor on B cells.

BACKGROUND

Toll-like receptors (TLRs) are pattern recognition receptors that bind avariety of microbial products such as microbial membrane lipids ornucleic acids. Antigen presenting cells (APC) including B cells, DCs andmacrophages express multiple TLRs that are bound by pathogens toactivate NF-κB and MAP kinase pathways, resulting in expression ofcostimulatory molecules and cytokine secretion. While TLRs are importantfor defense against infectious disease, increasing evidence suggeststhat TLRs also function as regulators of immune responses in cancer,autoimmune disease, and transplantation.

CD180 is a toll-like receptor (TLR) with a short cytoplasmic tail,lacking the Toll IL-1 Receptor (TIR) domain that mediates signaltransduction and that is shared by all other TLRs. CD180 has anextracellular structure analogous to TLR4, with 22 leucine rich repeatsand an associated co-receptor, MD1, required for CD180 expression. CD180is expressed at relatively high levels by B cells, and at lower levelsby dendritic cells (DC) and other myeloid cells.

G28-8 was the first mAb made to CD180, and was found to stimulateproliferation of B cells and to activate rapid calcium mobilization.Antibodies that bind to CD180 in the mouse also cause B cellproliferation and induce MAP kinase activation, and the responses dependupon the expression of the protein tyrosine kinases lyn and btk.However, the mechanism of CD180 signal transduction is unknown sinceCD180 does not appear to signal directly through its short cytoplasmictail.

CD180 is thought to be a regulator of TLR-4 responses. In CD180deficient animals, B cell proliferation and antibody responses to TLR-4stimulation by LPS were markedly reduced, whereas TNFα production andseptic shock after LPS treatment were increased. Thus CD180 has beenproposed to be a positive regulator of TLR-4 responses by B cells but anegative regulator of TLR-4 responses by myeloid cells and DCs. Therehave been no reported studies of the function of engineered CD180binding domains.

SUMMARY

In a first aspect, the present invention provides isolated CD180antibody or an antigen binding fragment thereof, wherein the antibody orantigen binding fragment thereof comprises a human CD180 binding domainlinked to an immunoglobulin constant region (Fc) domain that hasimpaired binding to human Fc receptor FcγRIIb.

In a second aspect, the present invention provides isolated nucleicacids encoding the antibodies of the invention.

In a third aspect, the present invention provides recombinant expressionvectors comprising the isolated nucleic acids of the invention.

In a fourth aspect, the present invention provides recombinant hostcells comprising the recombinant expression vectors of the invention.

In a fifth aspect, the present invention provides methods for producingmonoclonal immunoglobulin, comprising (a) culturing the host cells ofthe invention under conditions suitable for expression of thenucleic-acid encoded antibody; and (b) isolating the antibody from thecultured cells.

In a sixth aspect, the present invention provides methods for increasingserum immunoglobulin (Ig) level, comprising administering to a subjectin need thereof an antibody against CD180, or antigen binding fragmentthereof, wherein the antibody comprises a human CD180 binding domain anddoes not possess a functional Fc domain, wherein the antibody isadministered in an amount effective to increase serum Ig level.

In a seventh aspect, the present invention provides methods for plasmaprotein replacement therapy, comprising administering to a subject inneed thereof an antibody against CD180, or antigen binding fragmentthereof, wherein the antibody comprises a human CD180 binding domain anddoes not possess a functional Fc domain, wherein the antibody isadministered in an amount effective to maintain adequate antibody levelsin the subject.

In an eighth aspect, the present invention provides methods for treatinga disorder selected from the group consisting of an immune deficiency,hypogammaglobulinemia, hyper-IgM syndrome, autoimmune disease, cancer,graft rejection, and infections, comprising, administering to a subjectin need thereof an amount of an antibody against CD180, or antigenbinding fragment thereof, wherein the antibody comprises a human CD180binding domain and does not possess a functional Fc domain, wherein theamount of antibody administered is effective to treat immune deficiency,hypogammaglobulinemia, autoimmune disease, cancer, graft rejection, andinfections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SDS-PAGE analysis of G28-8 whole antibody and F(ab′)2.

FIG. 2 shows CFSE fluorescence intensity versus MHC class II (DR)brightness of human lymphocytes after incubation for 4 days with eithermedium alone, with mAb G28-8, or with F(ab′)2 fragment of G28-8.

FIG. 3 shows a histogram of CFSE fluorescence intensity versus cellfrequency gated on the DR positive population of human bloodlymphocytes, comparing the unstimulated, G28-8 stimulated, and G28-8F(ab′)2 stimulated cells.

FIG. 4 shows a dose titration of G28-8 or G28-8 F(ab′)2 in stimulationof human B cell proliferation, graphed as the percentage of DR positivelymphocytes proliferating after 4 days culture, calculated from FloJosoftware (Ashland, Oreg.).

FIG. 5 shows a schematic diagram of the structure of the CD180 specificscFv-Fc.

FIG. 6 shows the nucleotide (SEQ ID NO:1) and predicted amino acidsequence (SEQ ID NO:2) of an exemplary antibody of the invention, ascFv-Fc molecule constructed from the cloned variable regions of mAbG28-8. The Fe domain of the recombinant molecules is an altered humanIgG1 Fe domain with three amino acid changes (P238S, P331S, K322S) thatreduce the binding of the molecule to Fc-receptors and C1q.

FIG. 7 shows a Western Blot of three different G28-8 scFvs along withCTLA4Ig, and CTLA4-Ig-4-1BB fusion proteins. Each molecule wasimmunoprecipitated with protein A agarose from 0.5 ml culturesupernatants from COS7 cell transient transfections. COS7 cells weretransiently transfected by polyfect reagent (QIAGEN) using 2.5 ugplasmid minipreps.

FIG. 8 shows the binding of one of the recombinant G28-8 scFv-Fcmolecules to an immortalized human B cell line.

FIG. 9. Anti-CD180 rapidly induces Ig production independently of memoryrecall, T cell help, or MyD88 signals. A) WT mice received 250 μganti-CD180 or isotype control mAb, bled at indicated timepoints, andtotal serum Ig analyzed by ELISA. B) WT mice were immunized with 50 μgNP-CgG in alum, rested, and challenged with either 250 μg anti-CD180,100 μg anti-CD40 plus 10 μg LPS, 25 μg uncongugated CgG, 10 μg NP-CgG,or anti-CD180 plus NP-CgG, and bled at day 10 for NP-specific Iganalysis. Non-immunized mice for the naïve group were age matched. C)WT, TCR KO, and MyD88 KO mice were injected, bled at day 10, and totalserum Ig analyzed. p value between paired columns <0.001 unlessotherwise noted. n=4, replicates=4 for all experiments.

FIG. 10. T cell deficiency delays, but does not prevent,anti-CD180-induced Ig production. WT and TCR KO mice were injected with250 μg anti-CD180 or isotype control mAb and total serum Ig analyzed byELISA. p value between paired columns <0.001 unless otherwise noted.n=4, replicates=4.

FIG. 11. T-Independent Type-1 and 2, but not T-Dependent antigenspecific antibody, are decreased by co-administration of anti-CD180. A)WT mice were injected with 1 μg NP-LPS (0.7 NP/LPS) (TI-1), or 20 μgNP-Ficoll (152 NP/Ficoll) in combination with 250 μg anti-CD180 orisotype control mAb, bled on day 10, and serum analyzed for NP-specificantibody. B) WT and C) TCR KO mice were injected with 250 μg anti-CD180or isotype control mAb, and serum analyzed for anti-rat Ig-specific IgMand IgG2c antibody from day three, seven, and 10 time points. p valuebetween paired columns <0.001 unless otherwise noted. n=4, replicates=4for all experiments.

FIG. 12. Anti-CD180 injection expands and differentiates splenic B cellsin vivo. A) Spleens were harvested and cells enumerated three, seven,10, or 14 days following injection of 250 μg anti-CD180 or isotype mAb.Total splenocytes were subsetted by standard CD21/23/24 staining for Bcell subsets and CD3/4/8β staining for T cell subsets. n=3,replicates=3. B) T cell-deficient (TCR KO) and B cell-deficient (μMT)mice were injected and splenocytes analyzed at the day three timepointas in A. C) Unstimulated WT cells or those of a CD180 KO control werestained for CD180 expression and subsetted as in A.

FIG. 13. TLR signals reduce anti-CD180 induced Ig production but augmentproliferation in a MyD88-dependent manner. A) WT mice were injected withthe following TLR agonists in combination with either 250 μg anti-CD180or isotype control mAb: 1 μg LPS, 2 μg Pam₃CSK₄, or 10 μg CpG. Sera wereobtained at day ten and analyzed by ELISA. n=4, replicates=3. B) TLRligands LPS (10 μg), CpG (25 μg), or an equivalent volume of PBS wereco-injected with either anti-CD180 or isotype and splenocytes wereanalyzed at the day 3 timepoint as above. n=3, replicates=3. C) CFSElabeled splenocytes from TLR2/4 KO or WT mice were cultured withanti-CD180 (0.2 μg/ml, dashed line), LPS (0.5 μg/ml, grey fill), or both(solid line), with an unstimulated control (black fill). B cells weregated (FSC/SSC, B220⁺) and CFSE dilution analyzed. D) CFSE labeledsplenocytes from WT, TRIF KO, or MyD88 KO mice were cultured with gradeddoses of LPS alone or in combination with a constant 0.1 μg/ml dose ofanti-CD180. Proliferation Index is graphed against the corresponding LPSconcentration. n=1, replicates=3.

FIG. 14. Anti-CD180 synergizes for proliferation with all TLR ligandsthat signal through MyD88. A) Purified WT splenic B cells werestimulated with either TLR agonist alone, anti-CD180 alone, or both inconstant ratio combinations. Proliferation Index was calculated for eachseries and all curves graphed against the corresponding TLR agonistconcentrations. B) The three Proliferation Index curves were transformedinto a single Combination Index (CI) curve as described in “Materialsand methods”. Combination Index values of 1 (reference bar) indicatesimple additive effect (no interaction), CI values<1 indicate synergy(greater than additive effect), and CI values>1 indicateantagonism/inhibition. n=1, replicates=3.

FIG. 15. Anti-CD180 does not induce cytokine production by B cells butaugments induction by TLRs. A) Purified WT splenic B cells were seededat 1×10⁶ cells/ml in media with indicated stimulants. Overnight (24hour) culture supernatants were assayed by ELISA. B) Purified WT splenicDCs were treated as in A. Differences between paired columns are notsignificant unless otherwise noted. n=3, replicates=2.

DETAILED DESCRIPTION

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to.” Words using the singular or pluralnumber also include the plural or singular number, respectively.Additionally, the words “herein,” “above,” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of thisapplication. As used herein, the singular forms “a”, “an” and “the”include plural referents unless the context clearly dictates otherwise.“And” as used herein is interchangeably used with “or” unless expresslystated otherwise.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments.

All of the references cited herein are incorporated by reference.Aspects of the disclosure can be modified, if necessary, to employ thecompositions, functions and concepts of the above references andapplication to provide yet further embodiments of the disclosure. Theseand other changes can be made to the disclosure in light of the detaileddescription.

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure.

In a first aspect, the present invention provides isolated CD180antibody or an antigen binding fragment thereof, wherein the antibody orantigen binding fragment thereof comprises a human CD180 binding domainlinked to an immunoglobulin constant region (Fc) domain that hasimpaired binding to human Fc receptor FcγRIIb.

The inventors have discovered that CD180 activation by antibody bindingis inhibited by the antibody Fc domain's binding to the Fc receptorFcγRIIb. Thus, for example, a F(ab′)2 fragment of anti-CD180 mAb G28-8,specific for human CD180, was significantly more potent than the intactantibody in stimulation of proliferation of human peripheral blood Bcells. The inventors have further discovered that stimulation of CD180in vivo causes a large increase in serum immunoglobulin levels with norequirement for co-administration of adjuvants or TLR agonists. Thisantibody production capability of the antibodies of the invention iscompletely unexpected. Serum immunoglobulin is important for protectionfrom infection with bacteria, viruses, and parasites, and thus theantibodies and other therapeutics disclosed herein find application forthe limiting and/or treating a wide variety of infections.Immunosuppressed patients, including patients with cancer undergoingchemotherapy or bone marrow transplantation often have lowimmunoglobulin levels and are treated with immunoglobulin preparationscalled IVIg, which is also approved for use in patients with autoimmunedisease. Current methods for IVIg manufacturing use pooled serum fromlarge numbers of donors (>10,000/batch). Thus, the antibodies and othertherapeutics of the present invention represent a significantimprovement over current IVIg therapies, since CD180 stimulation causesendogenous B cells to differentiate and produce antibody, thus reducingthe potential for contamination with infectious agents and eliminatingthe need for infusions of large quantities of exogenous antibody. Theantibodies of the invention can be produced in mammalian cell lines,such as CHO cells, without requiring the addition of serum, and can alsobe expected to significantly reduce the cost of IVIg-equivalenttreatment.

The antibodies of the invention specifically bind to human CD180. Thephrase “specifically (or selectively) bind” to human CD180 or“specifically (or selectively) immunoreactive with,” human CD180, refersto a binding reaction that is determinative of the presence of CD180, ina heterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the antibodies bind to human CD180 atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to humanCD180, polymorphic variants, alleles, orthologs, and conservativelymodified variants, or splice variants, or portions thereof, can beselected to obtain only those antibodies that are specificallyimmunoreactive with human CD180 protein and not with other proteins.This selection may be achieved, for example, by subtracting outantibodies that cross-react with other molecules. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with human CD180. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies: ALaboratory Manual (1988) and Harlow & Lane, Using Antibodies (1999) fora description of immunoassay formats and conditions that can be used todetermine specific immunoreactivity).

In one embodiment, the antibodies of the invention compete with themonoclonal G28.8 antibody, or an antigen binding fragment thereof forbinding to human CD180. The G28-8 antibody is a well characterized mouseIgG1 monoclonal anti-CD180 antibody that is commercially available froma variety of sources (Santa Cruz Biotechnology, Inc. (CA); BDBiosciences (CA); etc.) As is well known to those of skill in the art, avariety of competition assays can be used to assess competition of theantibodies or other compounds of the invention with mAb G28.8, forbinding to human CD180. In one embodiment, competition also can beassessed by a flow cytometry test. For example, human CD180, orantigenic fragment thereof) can be incubated first with mAb G28.8, andthen with an antibody (or other therapeutic compound) of the presentinvention labeled with a detectable label (such as a fluorophore orbiotin). The test antibody typically is said to compete with thereference mAb G28.8 if the binding obtained after pre-incubation withsaturating amount of the mAb G28.8 is about 80% or less, such as about70% or less, about 60% or less, about 50% or less, about 40% or less,about 30% or less, about 20% or less, or about 10% or less, than thebinding obtained by the test antibody without pre-incubation with mAbG28.8. As will be clear to those of skill in the art, the same assaycould be conducted by pre-incubating with an antibody of the invention,and then labeling mAb G28.8 and carrying out the competition. Othercompetition assays are also well within the level of skill in the art,based on the teachings herein.

Binding affinity of the antibodies of the invention for human CD 180 istypically measured or determined by standard antibody-antigen assays,such as BIACORE™ competitive assays, saturation assays, or immunoassayssuch as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay(RIA). Such assays can be used to determine the dissociation constant ofthe antibody. The phrase “dissociation constant” refers to the affinityof an antibody for an antigen. Specificity of binding between anantibody and an antigen exists if the dissociation constant (K_(D)=1/K,where K is the affinity constant) of the antibody is <1 μM, preferably<100 nM, and most preferably <0.1 nM.

The antibodies of the invention have impaired binding to humanimmunoglobulin constant region Fc receptor FcγRIIb. The Fc domain in anantibody is composed of two heavy chains and is involved in selectivebinding to Fc receptors (FcR) on target cells. Surprisingly, theinventors have discovered that CD180 activation by anti-CD180 antibodybinding is inhibited by the antibody Fc domain. As used herein, theantibodies have “impaired binding to human immunoglobulin constantregion Fc receptor FcγRIIb” if binding of the Fc domain to theinhibitory Fc Receptor FcγRIIb is reduced by at least 50% relative tomAb G28.8 Fc domain binding to FcγRIIb. Methods for quantitating FcRbinding have been described in the art. For example, see Presta L G,“Engineering therapeutic antibodies for improved function” BiochemicalSociety Transactions 30 (part 4) 487-490 (2002), U.S. Pat. Nos.7,317,091; 7,662,925; 7,297,775, 7,371,826; 7,335,742; and 7,416,727,each incorporated by reference herein in their entirety. In variousembodiments, “impaired binding” means binding of the Fc domain to theinhibitory Fc Receptor FcγRIIb is reduced by at least 60%, 70%, 80%,90%, 100%, 200%, or more relative to mAb G28.8 Fc domain binding toFcγRIIb.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with human CD180, and includesmonoclonal antibodies. Various isotypes of antibodies exist, for exampleIgG1, IgG2, IgG3, IgG4, and other Ig, e.g., IgM, IgA, IgE isotypes. Theterm also includes genetically engineered forms such as chimericantibodies (e.g., humanized murine antibodies) and heteroconjugateantibodies (e.g., bispecific antibodies), and fully humanizedantibodies. As used throughout the application, the term “antibody”includes fragments with antigen-binding capability (e.g., Fab′, F(ab′)₂,Fab, Fv and rIgG. See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York (1998). The termalso refers to recombinant single chain Fv fragments (scFv). The termantibody also includes bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules aredescribed in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack andPluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra,Gruber et al. (1994) J Immunol: 5368, Zhu et al. (1997) Protein Sci6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) CancerRes. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301. Variousantigen binding domain-fusion proteins are also disclosed, e.g., in USpatent application Nos. 2003/0118592 and 2003/0133939, and areencompassed within the term “antibody” as used in this application.

An antibody immunologically reactive with human CD180 can be generatedby recombinant methods such as selection of libraries of recombinantantibodies in phage or similar vectors, see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); andVaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing ananimal with the antigen or with DNA encoding the antigen.

In one embodiment, the antibodies of the invention lack an Fc domainaltogether. Exemplary such antibodies include, but are not limited to aFab antibody, a Fab′ antibody, a (Fab′)₂ antibody, and an Fv antibody.In other embodiments, the antibodies of the invention possess an Fcdomain that has impaired binding to human immunoglobulin constant regionFc receptor FcγRIIb domain, whether through truncation or mutation, asdescribed herein. Such antibodies may be, for example, recombinant IgG,and an scFv-Fc antibodies.

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region, (theregions are also known as “domains”). Light and heavy chain variableregions contain four “framework” regions interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework regions and CDRs has beendefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

References to “V_(H)” or a “VH” refer to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, or Fab. References to “V_(L)” or a “VL” refer to thevariable region of an immunoglobulin light chain, including the lightchain of an Fv, scFv, dsFv or Fab. In one embodiment, the antibody or anantigen binding fragment of the present invention has one or more CDRsfrom mAb G28.8 (ie: 1, 2, or 3 of V_(H) CDR1, V_(H) CDR2, and V_(H)CDR3, and/or 1, 2, or 3 of V_(L) CDR1, V_(L) CDR2, and V_(L) CDR3.

The antibodies of the invention may be single chain Fv (“scFv”)antibodies. The phrase “single chain Fv” or “scFv” refers to an antibodyin which the variable domains of the heavy chain and of the light chainof a traditional antibody have been joined to form one chain. Typically,one or more linker peptides is inserted between the chains to allow forproper folding and creation of an active binding site.

The antibodies of the invention may be chimeric antibodies. A “chimericantibody” is an immunoglobulin molecule in which the constant region, ora portion thereof, is altered, replaced or exchanged so that the antigenbinding site (variable region) is linked to a constant region of adifferent or altered class, effector function and/or species, or anentirely different molecule which confers new properties to the chimericantibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.

The antibodies of the invention may be humanized antibodies. A“humanized antibody” is an immunoglobulin molecule which containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mAb G28.8 (as described herein). In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework (FR) regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody can alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin (Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Humanization can beessentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species.

The antibodies of the invention can also be human antibodies, which canbe produced using various techniques known in the art, including phagedisplay libraries (Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991);Marks et al., J. Mol. Biol. 222:581 (1991)). Other techniques are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boerneret al., J. Immunol. 147(1):86-95 (1991)). Similarly, human antibodiescan be made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg& Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

In some embodiments, the antibody of the invention is a single chain Fv(scFv). The V_(H) and the V_(L) regions of a scFv antibody comprise asingle chain which is folded to create an antigen binding site similarto that found in four chain antibodies. Once folded, noncovalentinteractions stabilize the single chain antibody. While the V_(H) andV_(L) regions of some antibody embodiments can be directly joinedtogether, one of skill will appreciate that the regions may be separatedby a peptide linker consisting of one or more amino acids. Peptidelinkers and their use are well-known in the art. See, e.g., Huston etal., Proc. Nat'l Acad. Sci. USA 8:5879 (1988); Bird et al., Science242:4236 (1988); Glockshuber et al., Biochemistry 29:1362 (1990); U.S.Pat. No. 4,946,778, U.S. Pat. No. 5,132,405 and Stemmer et al.,Biotechniques 14:256-265 (1993). Generally the peptide linker will haveno specific biological activity other than to join the regions or topreserve some minimum distance or other spatial relationship between theV_(H) and V_(L). However, the constituent amino acids of the peptidelinker may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity. Single chain Fv (scFv)antibodies optionally include a peptide linker. Methods of making scFvantibodies have been described. See, Huse et al., supra; Ward et al.supra; and Vaughan et al., supra.

In some embodiments, the antibodies are bispecific (or multi-specific)antibodies. Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least oneother antigen besides CD180 or that have binding specificities fordifferent CD180 epitopes. In one embodiment, the isolated antibody or anantigen binding fragment comprises one or more Fc domain mutations toimpair binding to the FcγRIIb receptor. Any suitable Fc domain mutantscan be used so that the resulting Fc domain binding to the FcγRIIbreceptor is reduced by at least 50% relative to mAb G28-8 Fc domainbinding to the FcγRIIb receptor. Fc mutations and truncations that canbe made to reduce binding of the antibodies of the invention to theFcγRIIb receptor can be made by those of skill in the art, based on theteachings herein, using binding assays to test for binding of theresulting antibodies to the FcγRIIb receptor as described herein and asare known in the art.

In one embodiment, the antibody is a human IgG4 mAb with human orhumanized variable regions. In another embodiment, the antibody has anFc domain of a human antibody with a mutation in the CH2 region so thatthe molecule is not glycosylated, including but not limited to N297 (EUnumbering for human IgG heavy chain constant region) in SEQ ID NO:6. Inanother embodiment, the Fc domain is human IgG1 with the three cysteinesof the hinge region (C220, C226, C229 in SEQ ID NO:6) each changed toserine, and the proline at position 238 of the CH2 domain changed toserine, and the proline at position 331 changed to serine. In anotherpreferred embodiment, the Fc domain is human IgG1 with N297 in SEQ IDNO:6 changed to any other amino acid. In another embodiment, the Fcdomain is human IgG1 with one or more amino acid change betweenpositions 292 and 300 in SEQ ID NO:6. In another embodiment, the Fcdomain is human IgG1 with an amino acid addition or deletion at anyposition between residues 292 and 300 in SEQ ID NO:6. In anotherembodiment, the Fc domain is human IgG1 with an SCC hinge; in a furtherembodiment an SSS hinge. In further embodiments, the Fc domain is humanIgG1 with an SCC hinge and P238S/P331S mutations; with an SCC hinge andP238S/K322S/P331S mutations; an SSS hinge and P238S/P331S mutations; oran SSS hinge and P238S/K322S/P331S mutations. In another embodiment, theFc domain is human IgG1 with mutations that alter binding by Fc gammareceptors (I, II, III) without affecting FcRn binding important for halflife. In further embodiments Fc domain is as disclosed in Ehrhardt andCooper, Curr. Top. Microbiol. Immunol, 2010 Aug. 3 (ImmunoregulatoryRoles for Fc Receptor-Like Molecules); Davis et al., Ann. Rev. Immunol,2007; 25:525-60 (Fc receptor-like molecules); and Swainson et al., J.Immunol. 2010 Apr. 1: 184(7):3639-47.

In certain embodiments, antibodies of the invention comprise an aminoacid substitution to an Fc domain which alters the antigen-independenteffector functions of the antibody, in particular the circulatinghalf-life of the antibody. Such antibodies exhibit either increased ordecreased binding to FcRn when compared to antibodies lacking thesesubstitutions and, therefore, have an increased or decreased half-lifein serum, respectively. Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, and such antibodies haveuseful applications in methods of treating mammals where long half-lifeof the administered antibody is desired. In contrast, Fc variants withdecreased FcRn binding affinity are expected to have shorter half-lives,and such antibodies are also useful, for example, for administration toa mammal where a shortened circulation time may be advantageous, e.g.where the starting antibody has toxic side effects when present in thecirculation for prolonged periods. Fc variants with decreased FcRnbinding affinity are also less likely to cross the placenta and, thus,are also useful in the treatment of diseases or disorders in pregnantwomen. In addition, other applications in which reduced FcRn bindingaffinity may be desired include those applications in which localizationthe brain, kidney, and/or liver is desired. In one exemplary embodiment,the antibodies of the invention exhibit reduced transport across theepithelium of kidney glomeruli from the vasculature. In anotherembodiment, the antibodies of the invention exhibit reduced transportacross the blood brain barrier (BBB) from the brain, into the vascularspace. In one embodiment, a hybrid nuclease antibody with altered FcRnbinding comprises at least one Fc domain (e.g., one or two Fc domains)having one or more amino acid substitutions within the “FcRn bindingloop” of an Fc domain. Exemplary amino acid substitutions which alteredFcRn binding activity are disclosed in International PCT Publication No.WO05/047327 which is incorporated by reference herein.

In other embodiments, an antibody of the invention comprises an Fcvariant comprising an amino acid substitution which alters theantigen-dependent effector functions of the polypeptide, in particularADCC or complement activation, e.g., as compared to a wild type Fcregion. In exemplary embodiment, said antibodies exhibit altered bindingto an Fc gamma receptor (e.g., CD16). Such antibodies exhibit eitherincreased or decreased binding to FcR gamma when compared to wild-typepolypeptides and, therefore, mediate enhanced or reduced effectorfunction, respectively. Fc variants with improved affinity for FcγRs areanticipated to enhance effector function, and such antibodies haveuseful applications in methods of treating mammals where target moleculedestruction is desired. In contrast, Fc variants with decreased FcγRbinding affinity are expected to reduce effector function, and suchantibodies are also useful, for example, for treatment of conditions inwhich target cell destruction is undesirable, e.g., where normal cellsmay express target molecules, or where chronic administration of theantibody might result in unwanted immune system activation. In oneembodiment, the antibody comprising an Fc exhibits at least one alteredantigen-dependent effector function selected from the group consistingof opsonization, phagocytosis, complement dependent cytotoxicity,antigen-dependent cellular cytotoxicity (ADCC), or effector cellmodulation as compared to a polypeptide comprising a wild type Fcregion.

In one embodiment the antibody exhibits altered binding to an activatingFcγR (e.g. FcγI, FcγIIa, or FcγRIIIa). In another embodiment, theantibody exhibits altered binding affinity to an inhibitory FcγR (e.g.FcγRIIb). Exemplary amino acid substitutions which altered FcR orcomplement binding activity are disclosed in International PCTPublication No. WO05/063815 which is incorporated by reference herein.

An antibody of the invention may also comprise an amino acidsubstitution which alters the glycosylation of the CD180 bindingmolecule. For example, the Fc domain of the antibody may comprise an Fcdomain having a mutation leading to reduced glycosylation (e.g., N- orO-linked glycosylation) or may comprise an altered glycoform of thewild-type Fc domain (e.g., a low fucose or fucose-free glycan). Inanother embodiment, the antibody has an amino acid substitution near orwithin a glycosylation motif, for example, an N-linked glycosylationmotif that contains the amino acid sequence NXT or NXS. Exemplary aminoacid substitutions which reduce or alter glycosylation are disclosed inInternational PCT Publication No. WO05/018572 and US Patent PublicationNo. 2007/0111281, which are incorporated by reference herein.

In a further embodiment, the Fc domain is a human IgG1 Fc domaincomprising amino acid changes in one or more of P238S, P331S, and K322S(including but not limited to P238S/P331S, K322S, and combinationsthereof). In this embodiment, it is further preferred that the antibodyis an scFv-Fc antibody, and it is further preferred that the antibodycomprises an amino acid sequence according to SEQ ID NO:2. As disclosedherein, the inventors have shown that such antibodies are significantlymore potent that intact antibody (with functional Fc domain) instimulation of proliferation of human peripheral blood B cells andproduction of serum immunoglobulin in mice.

As will be understood by those of skill in the art, in the antibodies ofany embodiment or combination of embodiments disclosed herein,insertions/linkers can be inserted between functional domains. In onenon-limiting embodiment, one or more linkers can be inserted betweenVL-VH or VH-VL; for example, ((gly4ser)3 and (gly4ser)4 insertions afterthe KLEIK (SEQ ID NO:9) sequence of the VL and before the beginning ofVH—in this case EVQ—or after the LTVSS (SEQ ID NO:10) of the VH andbefore the DIQ of the VL—depending on the orientation of the V regions)Linkers could also be inserted after the scFv and before the hinge orother domains in more complex molecules. These could be gly-ser typelinkers or other linkers, depending on the desired functional or spacingproperties required. Other linkers could be inserted after the CH3domain (PGK in human IgG1, to link the rest of a multispecific moleculeto another binding moiety, such as another scFv)

In another embodiment, a polypeptide linker comprises or consists of agly-ser linker. As used herein, the term “gly-ser linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser linker comprises an amino acid sequence of the formula(Gly₄Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). Apreferred gly/ser linker is (Gly₄Ser)4. Another preferred gly/ser linkeris (Gly₄Ser)3. Another preferred gly/ser linker is (Gly₄Ser)5. Incertain embodiments, the gly-ser linker may be inserted between twoother sequences of the polypeptide linker (e.g., any of the polypeptidelinker sequences described herein). In other embodiments, a gly-serlinker is attached at one or both ends of another sequence of thepolypeptide linker (e.g., any of the polypeptide linker sequencesdescribed herein). In yet other embodiments, two or more gly-ser linkerare incorporated in series in a polypeptide linker. In one embodiment, apolypeptide linker of the invention comprises at least a portion of anupper hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4molecule), at least a portion of a middle hinge region (e.g., derivedfrom an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of gly/seramino acid residues (e.g., a gly/ser linker such as (Gly₄Ser)n).

In another embodiment, a polypeptide linker of the invention comprises anon-naturally occurring immunoglobulin hinge region domain, e.g., ahinge region domain that is not naturally found in the polypeptidecomprising the hinge region domain and/or a hinge region domain that hasbeen altered so that it differs in amino acid sequence from a naturallyoccurring immunoglobulin hinge region domain. In one embodiment,mutations can be made to hinge region domains to make a polypeptidelinker of the invention. In one embodiment, a polypeptide linker of theinvention comprises a hinge domain which does not comprise a naturallyoccurring number of cysteines, i.e., the polypeptide linker compriseseither fewer cysteines or a greater number of cysteines than a naturallyoccurring hinge molecule.

It will be understood by those of skill in the art that these variousembodiments of the Fc mutations can be combined in the antibodies of theinvention, unless the context clearly indicates otherwise. Similarly, itwill be understood by those of skill in the art that these variousembodiments of the Fc mutations can be mutations to the Fc domain of mAbG28.8, unless the context clearly indicates otherwise.

The antibodies of the invention may comprise conservative amino acidsubstitutions. As to amino acid sequences, one of skill will recognizethat individual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles consistent with thedisclosure. Typically conservative substitutions for one another: 1)Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

The antibodies of the invention may comprise one or more amino acidresidues that are artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers, those containing modified residues, and non-naturallyoccurring amino acid polymer. The term “amino acid” refers to naturallyoccurring and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function similarly to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, e.g., an α carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functionssimilarly to a naturally occurring amino acid.

The antibodies of any embodiment of the invention can further beconjugated to an effector moiety. The effector moiety can be any numberof molecules, including labeling moieties such as radioactive labels orfluorescent labels, a TLR ligand or binding domain, or can be atherapeutic moiety. If the effector moiety is a therapeutic moiety, itwill typically be a cytotoxic agent. In this method, targeting thecytotoxic agent to cancer cells, results in direct killing of the targetcell. Cytotoxic agents are numerous and varied and include, but are notlimited to, cytotoxic drugs or toxins or active fragments of suchtoxins. Suitable toxins and their corresponding fragments include RNase,diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,curcin, crotin, phenomycin, enomycin, auristatin and the like. Cytotoxicagents also include radiochemicals made by conjugating radioisotopes toantibodies of the invention, or binding of a radionuclide to a chelatingagent that has been attached to the antibody.

In another embodiment, the antibodies of the invention are modified toextend half-life, such as by attaching at least one molecule to theantibody for extending serum half life, including but not limited to apolyethlyene glycol (PEG) group, serum albumin, transferrin, transferrinreceptor or the transferrin-binding portion thereof, or combinationsthereof. As used herein, the word “attached” refers to a covalently ornoncovalently conjugated substance. The conjugation may be by geneticengineering or by chemical means.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is isolated. In particular,an isolated nucleic acid is separated from some open reading frames thatnaturally flank the gene and encode proteins other than protein encodedby the gene. The term “isolated” in some embodiments denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Preferably, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure. In this sense, purification does notrequire that the purified compound be homogenous, e.g., 100% pure.

In another embodiment, the antibodies of the invention are present in apharmaceutical formulation. In this embodiment, the antibodies, orpharmaceutically acceptable salts thereof, are combined with apharmaceutically acceptable carrier. Suitable acids which are capable offorming such salts include inorganic acids such as hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid and the like; and organic acids such asformic acid, acetic acid, propionic acid, glycolic acid, lactic acid,pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid,fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonicacid, sulfanilic acid and the like. Suitable bases capable of formingsuch salts include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanol-amines (e.g., ethanolamine,diethanolamine and the like).

The pharmaceutical compositions of the invention may be made up in anysuitable formulation, preferably in formulations suitable foradministration by injection. In one embodiment, between about 50 andabout 500 mg of antibody are present in about 1 to about 2 ml of theformulation; preferably between about 100 mg and about 250 mg in about 1to about 2 ml of the formulation. In these embodiments, the antibody orantigen binding fragment thereof are present in about 25 mg/ml to about500 mg/ml; in further embodiments, at between about 50 mg/ml and 250mg/ml. Such pharmaceutical compositions can be used, for example, in themethods of the invention for increasing serum Ig levels or for plasmaprotein replacement therapy. The compositions of the invention are adramatic improvement over current methods, which involve lengthy IVinfusions of large amounts (150 grams or more) of suspended antibody.

In a second aspect, the present invention provides isolated nucleicacids encoding an antibody according to any embodiment of the presentinvention. The isolated nucleic acid sequence may comprise RNA or DNA.Such isolated nucleic acid sequences may comprise additional sequencesuseful for promoting expression and/or purification of the encodedprotein, including but not limited to polyA sequences, modified Kozaksequences, and sequences encoding epitope tags, export signals, andsecretory signals, nuclear localization signals, and plasma membranelocalization signals. In one preferred embodiment, the isolated nucleicacid encodes a polypeptide with an amino acid sequence according to SEQID NO:2. In another embodiment, the isolated nucleic acid comprises orconsists of the nucleic acid of SEQ ID NO:1, or a mRNA product thereof.The isolated nucleic acid sequences may further comprise conservativelymodified variants of these nucleotide sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or essentiallyidentical amino acid sequences, or where the nucleic acid does notencode an amino acid sequence, to essentially identical or associated,e.g., naturally contiguous, sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode most proteins. For instance, the codons GCA, GCC, GCG and GCU allencode the amino acid alanine. Thus, at every position where an alanineis specified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

In a third aspect, the present invention provides recombinant expressionvectors comprising the isolated nucleic acids of the invention. The term“recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, nucleic acid, proteinor vector, has been modified by the introduction of a heterologousnucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus,e.g., recombinant cells express genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under expressed or not expressed atall. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid, e.g., using polymerases and endonucleases, in a form notnormally found in nature. In this manner, operably linkage of differentsequences is achieved. Thus an isolated nucleic acid, in a linear form,or an expression vector formed in vitro by ligating DNA molecules thatare not normally joined, are both considered recombinant for thepurposes disclosed herein. It is understood that once a recombinantnucleic acid is made and reintroduced into a host cell or organism, itwill replicate non-recombinantly, i.e., using the in vivo cellularmachinery of the host cell rather than in vitro manipulations; however,such nucleic acids, once produced recombinantly, although subsequentlyreplicated non-recombinantly, are still considered recombinant for thepurposes disclosed herein. Similarly, a “recombinant protein” is aprotein made using recombinant techniques, i.e., through the expressionof a recombinant nucleic acid as depicted above.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any promoter capable of effectingexpression of the gene product. The promoter sequence used to driveexpression of the disclosed nucleic acid sequences in a mammalian systemmay be constitutive (driven by any of a variety of promoters, includingbut not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven byany of a number of inducible promoters including, but not limited to,tetracycline, ecdysone, steroid-responsive). The construction ofexpression vectors for use in transfecting prokaryotic cells is alsowell known in the art, and thus can be accomplished via standardtechniques. (See, for example, Sambrook, Fritsch, and Maniatis, in:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.). The expression vector must be replicablein the host organisms either as an episome or by integration into hostchromosomal DNA. In a preferred embodiment, the expression vectorcomprises a plasmid. However, the invention is intended to include otherexpression vectors that serve equivalent functions, such as viralvectors.

In a fourth aspect, the present invention provides recombinant hostcells comprising the recombinant expression vectors of the invention.The host cells can be either prokaryotic or eukaryotic. The cells can betransiently or stably transfected. Such transfection of expressionvectors into prokaryotic and eukaryotic cells (including but not limitedto Chinese hamster ovary (CHO) cells) can be accomplished via anytechnique known in the art, including but not limited to standardbacterial transformations, calcium phosphate co-precipitation,electroporation, or liposome mediated-, DEAE dextran mediated-,polycationic mediated-, or viral mediated transfection. (See, forexample, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manualof Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. NewYork, N.Y.).

In a fifth aspect, the present invention provides methods for method forproducing immunoglobulin, comprising (a) culturing the host cells of theinvention under conditions suitable for expression of the nucleic-acidencoded antibody; and (b) isolating the antibody from the culturedcells. In this embodiment, any embodiment of the host cells of theinvention can be used. In one embodiment, prokaryotic host cells areused and the serum Ig is produced by methods known in the art, includingbut not limited that disclosed in US Published Patent Application20110003337. In another embodiment, mammalian host cells are used, andstandard culture conditions and isolation techniques are used.

Serum immunoglobulin is important for protection from infection withbacteria, viruses, and parasites, and thus the antibodies and othertherapeutics disclosed herein find application for the limiting and/ortreating a wide variety of infections. Immunosuppressed patients,including patients with cancer undergoing chemotherapy or bone marrowtransplantation often have low immunoglobulin levels and are treatedwith immunoglobulin preparations called IVIg, which is also approved foruse in patients with autoimmune disease. Current methods for IVIgmanufacturing use pooled serum from large numbers of donors(>10,000/batch), thus increasing risk of infection, and requiresinfusion of 150+ grams of purified antibody over a two hour span. Thus,the methods of this aspect of the present invention represent asignificant improvement over current IVIg production techniques, in thatmuch smaller amounts of antibody can be administered intramuscularly orintravenuously, which stimulate the subject's B cells to produce largeamounts of serum antibody and greatly reduces the risk of infection.

In a sixth aspect, the present invention provides methods for increasingserum immunoglobulin (Ig) level, comprising administering to a subjectin need thereof an antibody against CD180, or antigen binding fragmentthereof, wherein the antibody does not possess a functional Fc domain,wherein the antibody is administered in an amount effective to increaseserum Ig level.

In a seventh aspect, the present invention provides methods for plasmaprotein replacement therapy, comprising administering to a subject inneed thereof an antibody against CD180, or antigen binding fragmentthereof, wherein the antibody does not possess a functional Fc domain,wherein the antibody is administered in an amount effective to maintainadequate antibody levels in the subject.

In the methods of the invention, the antibodies “do not possess afunctional Fc domain,” meaning that the Fc domain has impaired bindingto immunoglobulin constant region Fc receptor FcγRIIb, as describedherein. Thus, antibodies for use in the methods of the invention can beany of those disclosed herein, including antibodies that lack an Fcdomain, and those in which the Fc domain is mutated or truncated toimpair binding to FcγRIIb.

Most immunoglobulin present in the blood is natural antibody thatcontains germ-line variable regions without extensive somatic mutation.The natural antibody has reactivity towards multiple pathogens includingbacteria and viruses, and is important in first-line defense andprotection from infection. Natural antibodies mediate directneutralization of bacteria or viruses present in the circulation andprotect mice from encephalitis induced by vesicular stomatitis virus(VSV). In addition, natural antibodies have protective activity againstintravenous infection with S. pneumonia. Natural antibodies alsocontribute to protection by enhancing phagocytosis of parasites. Resultsobtained in mice deficient in secreted IgM demonstrated a protectiverole for natural antibodies in bacterial clearance from the peritonealcavity via activation of the complement cascade and the formation of thelytic complex, and also in the protection against a mucosal influenzainfection. Natural antibodies provide an important link between innateand adaptive immunity by binding complement components in immunecomplexes, thus enhancing the uptake of antigen by complement receptors.Administration of the antibodies and other therapeutic moleculesdisclosed herein to subjects (e.g., humans, other non-human mammals)will increase levels of antibodies similar to natural antibody andshould thus provide protection and enhanced immunity to a broad range ofviruses, parasites, and bacteria. CD180 stimulation with embodiments ofantibodies and other therapeutic molecules disclosed herein shouldenhance innate protection from multiple environmental toxins andinfectious agents.

The sixth and seventh aspects of the invention can be used with anysubject that can benefit from increased Ig levels and/or plasma proteinreplacement therapy. In a preferred embodiment, the subject's immunesystem is dysregulated (ie: overactive or deficient) due to a disorder,disease, or treatment including but not limited to subjects with IgGdeficiencies, hypogammaglobulinemia, other immune deficiencies,undergoing chemotherapy or radiation therapy (ex: cancer patients, bonemarrow transplantation patients, etc.), infections (bacterial, viral,parasite), autoimmune diseases, and hyper IgM syndrome.

In an eighth aspect, the present invention provides methods for treatinga disorder selected from the group consisting of an immune deficiency,hypogammaglobulinemia, autoimmune disease, cancer, graft rejection, andinfections, comprising, administering to a subject in need thereof anamount of an antibody against CD180, or antigen binding fragmentthereof, wherein the antibody does not possess a functional Fc domain,wherein the amount of antibody administered is effective to treat immunedeficiency, hypogammaglobulinemia, autoimmune disease, cancer, graftrejection, or infection. In addition to the methods disclosed above, theantibodies and other therapeutic molecules described herein are alsouseful for therapy of patients with autoimmune disease, cancer, or withdysregulated immune systems. As used herein, “treat” or “treating” meansaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting or preventing development of symptomscharacteristic of the disorder(s) being treated (ex: immune deficienciesin cancer patients (or other patients) undergoing chemotherapy and/orradiation therapy); (c) inhibiting worsening of symptoms characteristicof the disorder(s) being treated; (d) limiting or preventing recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting or preventing recurrence of symptoms in patients thatwere previously symptomatic for the disorder(s).

In each of the sixth, seventh, and eighth aspects of the invention, theantibody to be administered can be according to any embodiment orcombination of embodiments of the antibodies disclosed herein. Thus, inone embodiment of any of these aspects, the antibody or antigen bindingfragment thereof is the antibody or antigen binding fragment is selectedfrom the group consisting of a Fab antibody, a Fab′ antibody, a (Fab′)₂antibody, a rIg, and an scFv-Fc antibody. In another embodiment, theantibody or an antigen binding fragment has one or morecomplementarity-determining regions from mAb G28.8. In a furtherembodiment, the antibody comprises one or more Fc domain mutations toimpair binding the CD180. In a still further embodiment, the Fc domainis a human IgG1 Fc domain comprising amino acid changes P238S, P331S,and K322S. In another embodiment, the antibody is an scFv-Fc antibody.In yet another embodiment, the antibody comprises an amino acid sequenceaccording to SEQ ID NO:2. In any of these embodiments, the antibodiescan be modified to extend half-life, such as by attaching at least onemolecule to the antibody for extending serum half life, including butnot limited to a polyethlyene glycol (PEG) group, serum albumin,transferrin, transferrin receptor or the transferrin-binding portionthereof, or combinations thereof.

Any suitable subject capable of immunoglobulin production can be treatedaccording to the methods of the invention, including but not limited tohumans, primates, dogs, cats, cattle, etc. The antibody to be used would(a) be specific for a CD180 of the subject to be treated (ie: humanCD180 specific for human subjects, etc.), and (b) have a non-functionalFc domain that has impaired binding to FcγRIIb of the subject to betreated.

As used herein for all of the methods of the invention, an “amounteffective” of the one or more antibodies or other therapeutics is anamount that is sufficient to provide the intended benefit of treatment.An effective amount of the antibodies or other therapeutics that can beemployed ranges generally between about 0.01 μg/kg body weight and about10 mg/kg body weight, preferably ranging between about 0.05 μg/kg andabout 5 mg/kg body weight. However dosage levels are based on a varietyof factors, including the type of injury, the age, weight, sex, medicalcondition of the individual, the severity of the condition, the route ofadministration, and the particular compound employed. In one embodiment,between about 50 and about 500 mg of antibody are administered IM or IVin about a 1 ml to about a 2 ml formulation; preferably between about100 mg and about 250 mg in about a 1 ml to about a 2 ml formulation.Frequency of administration can also be determined by an attendingphysician in light of all appropriate factors. In exemplary embodiments,administration is one-two time per day, every other day, one-two timesper week, once per month, once every other month, etc.

For administration, the antibodies or other therapeutics are ordinarilycombined with one or more excipients appropriate for the indicated routeof administration. The compounds may be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulphuric acids, acacia, gelatin, sodium alginate,polyvinylpyrrolidine, dextran sulfate, heparin-containing gels, and/orpolyvinyl alcohol, and tableted or encapsulated for conventionaladministration. Alternatively, the antibodies or other therapeutics maybe dissolved in saline, water, polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, and/or various buffers. Theantibodies may be linked to other compounds to promote an increasedhalf-life in vivo, such as described herein. Such linkage can becovalent or noncovalent as is understood by those of skill in the art.

The antibodies or other therapeutics may be administered by any suitableroute, preferably intravenous or intramuscular administration.

In a ninth aspect, the present invention provides methods foridentifying candidate immunostimulatory compounds comprising identifyingmolecules that (a) bind to and stimulate CD180, and B) have minimalbinding (less than that of a normal antibody) to the inhibitory Fcreceptor FcγRIIb on B cells. CD180 binding can be measured with any sortof capture assay, such as cell based flow cytometry assays or capture toan ELISA plate coated with purified soluble CD180 protein and thendetected with an anti-Ig second step. Modifications to Fc domains toreduce binding to Fc receptors are disclosed herein and are known in theart. End activity of the constructs versus parent wild type antibodies(such as G28-8) can be carried out using any suitable technique,including dye-dilution proliferation assays. In one exemplaryembodiment, B cells (such as PBMCs) can be labeled with a fluorophore(such as CFSE, etc.) cultured for a suitable period of time (such as 3-4days) with additions of the compounds to be screened (a first cellpopulation for the constructs to be tested, and a second population tobe treated with control antibody), and fluorescence of the cells on eachpopulation assessed at the end point. As the fluorescent dye is halvedas each cell division occurs the culture with the least fluorescence hadthe most active stimulant.

EXAMPLE 1

F(ab′)2 fragments of mAb G28-8 were generated by pepsin digestion usingthe method of Parham (J Immunol. 1983 December; 131(6):2895-902). Thepepsin digestion of G28-8 was complete, since the mAb was 160 kDa priorto digestion, and no residual 160 kDa undigested protein was detectedafter pepsin treatment. The digested protein was mostly 100 kDa F(ab′)2fragment as shown on the SDS PAGE analysis of nonreduced protein, withsmaller digestion fragments of approximately 75 kDa and 45 kDa alsopresent (FIG. 1).

FIG. 2 shows CFSE fluorescence intensity versus MHC class II (DR)brightness of lymphocytes after incubation for 4 days with either mediumalone, with mAb G28-8, or with F(ab′)2 fragment of G28-8. Humanperipheral blood mononuclear cells (PBMC) were isolated bycentrifugation of heparanized peripheral blood, diluted 1:1 withphosphate buffered saline, over Lymphocyte Separation Medium (LSM,Cappel, Aurora, Ohio) according to manufacturer's instructions. PBMCwere labeled with carboxyfluorescein diacetate, succinimidyl ester(CFSE, Molecular Probes, Oregon) according to the method of Lyons et al(J. Immunol. Methods, 171:131-137, 1994) and cultured in RPMI mediasupplemented with glutamine, P/S, pyruvate, and 10% FBS. Cells werecultured with no added antibody, with mAb G28-8, or with F(ab′)2fragment of G28-8 as indicated. After 4 days of culture at 37 degrees in5% CO2, cells were stained with PE-labeled antibody to MHC class II (DR)(BD-Pharmingen 555812, lot28211) and analyzed by flow cytometry.Lymphocytes were analyzed using a FACS™ SCAN (BD, Mountain View,Calif.), and data was analyzed using FLOWJO™ software (Ashland, Oreg.).Example 1 shows that removal of the Fc region of CD 180 mAb G28-8 bypepsin digestion increased its potency in stimulation of B cellproliferation, and that the increased potency was seen as an increase inthe maximum effect rather than simply a shifting of the dose responsecurve. Example 1 also shows the sequence and binding of a recombinantscFv-Fc molecules constructed from the cloned variable regions of mAbG28-8, and their binding to human B cells.

FIG. 3 shows a histogram of CFSE fluorescence intensity versus cellfrequency gated on the DR positive population of blood lymphocytes,comparing the unstimulated, G28-8 stimulated, and G28-8 F(ab′)2stimulated cells. FIG. 4 shows a dose titration of G28-8 or G28-8F(ab′)2 in stimulation of B cell proliferation, graphed as thepercentage of DR positive lymphocytes proliferating after 4 daysculture, calculated from FLOWJO™ software (Ashland, Oreg.). FIG. 5 showsa schematic diagram of the structure of the CD180 specific scFv-Fc. FIG.6 shows the nucleotide and predicted amino acid sequence of a scFv-Fcmolecule constructed from the cloned variable regions of mAb G28-8. TheFe domain of the recombinant molecules is an altered human IgG1 Fedomain with three amino acid changes (P238S, P331S, K322S) that reducethe binding of the molecule to Fc-receptors and C1q. FIG. 7 shows aWestern Blot of three different G28-8 scFvs along with CTLA4Ig, andCTLA4-Ig-4-1BB fusion proteins. Each molecule was immunoprecipitatedwith protein A agarose from 0.5 ml culture supernatants from COS7 celltransient transfections. COS7 cells were transiently transfected byPOLYFECT™ reagent (QIAGEN) using 2.5 ug plasmid minipreps. FIG. 8 showsthe binding of one of the recombinant G28-8 scFv-Fc molecules to human Bcells.

To prepare an scFv, total RNA was isolated from hybridoma cells growingin log phase using QIAGEN RNEASY™ kits, including QIA shredder columnpurification to homogenize cell lysates, and purification over RNEASY™mini-columns. Then cDNA was synthesized and anchor tailed, and PCRperformed using an anchor-tail (CCCCCC)-complementary primer and aprimer, which anneals specifically to the antisense strand of theconstant region of either mouse (or rat) Ck or mouse CH 1 (or rat) forthe appropriate isotype. The amplified variable region fragments werethen TOPO® cloned (Invitrogen), and clones with inserts of the correctsize sequenced. Consensus sequence for each variable domain aredetermined from sequences of at least 4 independent clones. The scFv isconstructed by insertion of a variable length (gly4ser) linker (10-20amino acids) or by PCR using overlapping primers containing a synthetic(gly4ser)3, a (gly4ser)4 or a hydrophilic linker containing a (glyser)motif at each end, with the linker domain inserted between the VL and VHregions. Constructs are fused to the human VK3 light chain signalpeptide or alternatively a synthetic leader sequence derived from thesequences of several different leaders, with a Kozak signal sequenceincluded to improve expression level of the expressed fusion proteins.Small cassettes were designed which incorporated a 5′ restriction site,Kozak consensus sequence, and the leader peptide from the human VK3germline sequence which includes an in frame Age I site at the end ofthe leader peptide, just upstream of the framework region for the lightchain variable domain. Overlapping, partially complementaryoligonucleotides were used in PCR extension reactions to create theseshort cassettes. PCR products of the correct size were isolated by gelelectrophoresis and cloned into the pCR®-2.1 TOPO® (Invitrogen) vector.

PCR reactions were performed on the TOPO® cloned DNA using a 30 cycleprogram with the following profile: 94 C, 30 sec; 55 C, 30-60 sec; 68 C,30-60 sec, followed by a final extension at 72 C for 8 minutes. PCRproducts were gel purified and fragments recovered using a QIAQUICK™ gelextraction kit (QIAGEN, Valencia, Calif.). Fragments were subcloned intoPuc derived vectors for creating fusion genes and assembly of thescFv-Fc construct.

Usually, a VH was subcloned into a particular vector with the leaderpeptide cassette as a three way ligation, positive clones identified byrestriction digestion, and these clones recut with the appropriateenzymes for inserting linker and VL domains together as a secondthree-way ligation. Fragments were mixed together in the appropriateratios to optimize three-way ligation reactions.

Fragments were diluted 1:50 and 1 microliter used for overlap extensionPCR reactions when linkers were attached in this manner instead of bymulti-fragment ligation. Diluted overlapping PCR products were added tostandard PCR reactions without additional primers and run for 2 cycles.Cycling was then paused, and the flanking VL 5′ and VH 3′ primers wereadded. Cycling was resumed and allowed to complete the remainder of the30 cycle program. The temperature profile was 94 C, 60 sec; 55 C, 60sec; and 68 C, 60 sec for 30 cycles. Human Fc domains (hinge, CH2, andCH3 domains) were isolated from blood (human and macaque) or splenic(mouse) RNA. Sequences were altered using overlap extension PCR tointroduce mutations at residues implicated in mediating ADCC and CDCeffector functions.

Initial expression studies were performed by transient transfection ofCOS cells, using POLYFECT™ (QIAGEN, Valencia, Calif.) transfectionreagent according to manufacturer's instructions. Culture supernatantswere harvested at 72 hours posttransfection and screened for binding tothe Ramos, Bjab, or T51 B cell lymphoma lines which expresses highlevels of varying levels of human CD180. Constructs were thentransfected into CHO DG44 cells to create stable cell lines as describedpreviously.

Stable production of the −Ig fusion protein was achieved byelectroporation of a selectable, amplifiable plasmid, pDG, containingthe RNase-Ig cDNA under the control of the CMV promoter, into ChineseHamster Ovary (CHO) cells. The pDG vector is a modified version ofpcDNA3 encoding the DHFR selectable marker with an attenuated promoterto increase selection pressure for the plasmid. Plasmid DNA was preparedusing Qiagen maxiprep kits, and purified plasmid was linearized at aunique AscI site prior to phenol extraction and ethanol precipitation.Salmon sperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as carrierDNA, and 100 μg each of plasmid and carrier DNA was used to transfect10⁷ CHO DG44 cells by electroporation. Cells were grown to logarithmicphase in EX-CELL® 302 media (JRH Biosciences) containing glutamine (4mM), pyruvate, recombinant insulin, penicillin-streptomycin, and 2×DMEMnonessential amino acids (all from Life Technologies, Gaithersburg,Md.), hereafter referred to as “EX-CELL® 302 complete” media. Media foruntransfected cells also contained HT (diluted from a 100× solution ofhypoxanthine and thymidine) (Invitrogen/Life Technologies). Media fortransfections under selection contained varying levels of methotrexate(SigmaAldrich) as selective agent, ranging from 50 nM to 1 μM.Electroporations were performed at 280 volts, 950 microFarads.Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 125 cells/well to 2000cells/well. Culture media for cell cloning was EX-CELL® 302 complete,containing 50 nM methotrexate. Once clonal outgrowth was sufficient,serial dilutions of culture supernatants from master wells were screenedfor expression of −Ig fusion protein by use of an −IgG sandwich ELISA.Briefly, NUNC IMMULON™ II plates were coated overnight at 4° C. with 7.5microgram/ml F(ab′2) goat anti-mouse IgG (KPL Labs, Gaithersburg, Md.)in PBS. Plates were blocked in PBS/3% BSA, and serial dilutions ofculture supernatants incubated at room temperature for 2-3 hours. Plateswere washed three times in PBS/0.05% Tween 20, and incubated withhorseradish peroxidase conjugated F(ab′2) goat anti-human IgG(SouthernBiotechnologies), each at 1:3000 in PBS/1.0% BSA, for 1-2 hoursat room temperature. Plates were washed four times in PBS/0.05% Tween20, and binding detected with SUREBLUE RESERVE™, TMB substrate (KPLLabs, Gaithersburg, Md.). Reactions were stopped by addition of equalvolume of 1N HCl, and plates read at 450 nM. The clones with the highestproduction of the fusion protein were expanded into T25 and then T75flasks to provide adequate numbers of cells for freezing and for scalingup production of the fusion protein. Production levels were furtherincreased in cultures from the four best clones by progressiveamplification in methotrexate containing culture media. At eachsuccessive passage of cells, the EX-CELL® 302 complete media containedan increased concentration of methotrexate, such that only the cellsthat amplified the DHFR plasmid could survive. The production level ofthe top four unamplified master wells from the G28-8 scFvIg CHOtransfectants ranged from 30-50 micrograms/ml culture.

Supernatants were collected from CHO cells expressing the G28-8 scFvIgfiltered through 0.2 μm PES express filters (Nalgene, Rochester, N.Y.)and were passed over a Protein A-agarose (IPA 300 crosslinked agarose)column (Repligen, Needham, Mass.). The column was washed with columnwash buffer (90 mM Tris-Base, 150 mM NaCl, 0.05% sodium azide, pH 8.7)and bound protein was eluted using 0.1 M citrate buffer, pH 3.4.Fractions were collected into 1M bicarbonate buffer and proteinconcentration was determined at 280 nM using a NANODROP™ (WilmingtonDel.) microsample spectrophotometer, and blank determination using 0.1 Mcitrate buffer, pH 3.4 with 100 ul 1M bicarbonate buffer.

EXAMPLE 2

While current anti-human CD180 antibodies (prior to the presentinvention) are mouse IgG1 and bind fairly strongly to human Fcreceptors, generating a mixed signal to bound B cells, the rat IgG2aanti-mouse CD180 antibody used in Example 2 has very low affinity formouse Fc receptors, thus making it a natural mimetic for the non-FcRbinding constructs disclosed in the present application.

Materials and Methods

Mice

WT (C57BL/6), CD40 KO, B cell-deficient (μMT), and T cell-deficient(TCRβ/δ KO, TCR KO) mice were from Jackson Laboratory (Bar Harbor, Me.)and all other strains were on this background. MyD88 KO mice were giftsfrom K. Elkon (University of Washington, Seattle, Wash.). CD180 KO micewere a gift from C. Karp (Children's Research Foundation, Cincinnati,Ohio). All mice were sex and age matched and used at six to twelve weeksof age, except memory recall studies that utilized 60-week-old mice. Allinjections and immunizations were intraperitoneal with a fixed volume of200 μl in PBS. The University of Washington Institutional Animal Careand Use Committee approved all animal work.

Cell Preparation and Culture

Spleens were processed by LIBERASE™ (Roche, Indianapolis, Ind.)digestion for DCs or mechanical disruption. Erythrocytes were depletedby Gey's lysis for total splenocyte preparations. B cells or DCs wereisolated by three rounds of enrichment (STEM CELL technologies,Vancouver, BC, Canada) and purity exceeded 99% without expression ofactivation markers (CD69 or CD86) after 24 hours in unstimulatedcultures.

Total splenocytes or purified cells were cultured in complete medium(RPMI-1640 supplemented with 10% fetal calf serum [Hyclone, Logan,Utah], 4 mM glutamine, 1 mM Na pyruvate, 1× Non-Essential Amino Acids,100 IU/ml penicillin-streptomycin [Invitrogen, Carlsbad, Calif.], and 50uM 2-mercaptoethanol [Sigma-Aldrich, St. Louis, Mo.]) in the presence ofstimuli at a final cell density of 1×10⁶/mL for 64 hours at 37° C.

ELISA Measurement of Serum Antibody and In Vitro Cytokine Production

Sera were obtained after injection of mice with mAbs and/or TLRagonists. Polystyrene plates were coated with donkey anti-mouse IgG, orgoat anti-mouse IgM F(ab′)₂ (Jackson ImmunoResearch, West Grove, Pa.).After blocking with 4% nonfat dry milk in PBS-Tween, serial dilutions ofserum were added. Abs were detected with isotype-specific HRP conjugates(anti-IgG1, anti-IgG2b, and anti-IgG3 from ICL, Newberg, Oreg.; anti-IgMand anti-IgG2c from Southern Biotech, Birmingham, Ala.) and absorbancewas compared with standard curves generated from mouse monoclonalstandards (IgG3 from BioLegend, San Diego, Calif.; IgM from JacksonImmunoResearch; IgG2c from Southern Biotech; IgG1 and IgG2b standardswere purified in our laboratory) for absolute quantitation. Nocross-reactions between standards for the IgG subclasses, IgM, or theinjected Rat IgG2a mAbs were observed. Antigen specific antibody from4-hydroxy-3-nitro-phenacetyl (NP) conjugated LPS, NP-Ficoll, orNP-chicken gamma globulin (CgG) was captured with NP-BSA coated plates(all NP reagents from BioSearch Technologies, Inc., Novato, Calif.).

IL-6, IL-10, and TNF-α concentrations in 24-hour supernatants fromcultures of purified cells were measured by ELISA (DuoSets from R&DSystems, Minneapolis, Minn.) per the manufacturer's instructions.

Analysis of Lymphocyte Subsets and Proliferation

Flow cytometry analyses were performed on either a standard FACS™ SCANor FACSCanto (Becton Dickinson, Franklin Lakes, N.J.). Minimums of30,000 cells of the final gated population were used for all analyses.Data analysis was performed with FLOWJO™ (Tree Star, Ashland, Oreg.)software. Staining was performed for: CD3 and CD24 (Becton Dickinsonclones 145-2c11 and MI/69); CD4, CD8β, CD19, CD21, and CD23 (BioLegendclones RM4-5, YTS156.7.7, 6D5, 7E9, and B3B4); CDS, CD45R/B220, and CD86(Clones 53-7.3, RA3-6B2, and GL1 from eBioscience, San Diego, Calif.).Mouse anti-rat IgG secondary antibody was from Jackson ImmunoResearch.

CFSE (Invitrogen) labeling of cells was performed with a finalconcentration of 0.8 μM CFSE and 1.6×10⁷ cells/ml in 37° C. PBS for fourminutes. Proliferation Index was calculated by dividing the MFI forgated live singlet unstimulated B cells by the MFI of equivalently gatedcells from the stimulated sample. This measurement simultaneouslycaptures both percent proliferating cells and the average number ofdivisions per cell. A Proliferation Index of 1 indicates equivalence tounstimulated culture.

Synergy Determinations and Calculation of the Combination Index

The Combination Index (CI), a quantitative definition of synergy orantagonism, was calculated by the method of Chou and Talalay²¹ throughthe use of CalcuSyn software (Biosoft, Cambridge, United Kingdom). Asthe CI method is based on the median effect principle of the mass actionlaw, it is mechanism-independent.

Other Antibodies and Reagents

The anti-CD180 (RP/14) hybridoma was a gift from K. Miyake (Universityof Tokyo, Tokyo, Japan) and the rat IgG2a isotype control (9D6)hybridoma was a gift from R. Mittler (Emory University, Atlanta, Ga.).To ensure equivalence these mAb were sequentially purified on the sameprotein G column and routinely bioassayed both alone and in combinationwith polymyxin B sulfate. LPS (L2143) was from Sigma-Aldrich. SyntheticTLR agonists Pam₂CSK₄, Pam₃CSK₄, CL097, and CpG ODN1826 were fromInvivoGen (San Diego, Calif.).

Statistical Analyses

Raw data of experimental groups were analyzed either by one-way ANOVAfollowed by Bonferroni's Multiple Comparison Test (GraphPadPrismsoftware, version 4.0a for Macintosh, San Diego, Calif.) or bytwo-tailed, type two Student's t-test. Columnar data are represented asmean±standard error (SEM). A value of p<0.05 was considered to bestatistically significant and assigned *, while p<0.01 and p<0.001 wereassigned ** and ***, respectively.

Results

Anti-CD180 Injection Induces Polyclonal Ig Production of MultipleIsotypes

Because CD180 KO mice have low serum concentrations of IgG3¹¹, weexamined Ig concentrations of WT mice at 3, 7, 10, and 14 days afterinjection with either anti-CD180 or isotype-matched control mAb (theanti-CD180 antibody is an agonistic rat IgG2a that was not expected todeplete target cells or bind significantly to FcγRIIb). At no point didthe anti-CD180-injected mice show any evidence of distress, unlike TLR4agonists which rapidly induce septic shock.

Anti-CD180 alone increased serum Ig concentration of nearly everyisotype and subclass by day 3, with consistently rapid and dramaticincreases for IgG1, IgG2c, and IgG3 (12, 9.5, and 56-fold increase atday 10, respectively), while changes in serum concentration of IgM weremodest and IgG2b varied with an average of 1.5-fold reductions (FIG. 9Aand data not shown). Semi-quantitative immunoblots for IgA and IgE fromday 10 bleeds indicate that IgA concentrations were equivalent incontrol and anti-CD180 treated mice, while IgE concentrations increasedroughly 10-fold (data not shown).

We examined whether rapid production of IgM and IgG1 after CD180stimulation was due to reactivation of memory B cells. WT mice wereimmunized with NP-CgG in alum and rested for 50 weeks before injectionof recall stimuli. While recall Ag administration without adjuvantproduced robust NP-specific IgG1, neither anti-CD180 nor inflammatorystimuli (LPS plus anti-CD40) induced significant recall compared tounconjugated CgG (FIG. 9B). Addition of anti-CD180 stimulation with Agdid not significantly impact recall IgG1 responses.

Anti-CD180-induced Ig production required neither T cells, CD40 signalsupport, nor TLR signaling as the increase in IgG concentrations stilloccurred after injection of TCR KO, CD40 KO, and MyD88 KO mice (FIG.9C). IgM production was largely bypassed and IgG production wasstrikingly delayed in TCR KO mice, indicating a supportive role for Tcells despite dispensability for the overall anti-CD180-induced Igeffect (FIG. 10).

To assess whether anti-CD180-induced Ig is polyclonal or an exuberantAg-specific response, we examined both the effect of co-administrationof anti-CD180 with model T cell-independent (TI) antigens as well as Igproduced against the rat IgG2a anti-CD180 mAb itself (FIG. 11).Ag-specific antibody was reduced or unchanged for all isotypes with bothT cell-independent type 1 (TI-1, intrinsic B cell activating Ag, NP-LPS)and type 2 (TI-2, polyvalent Ag, NP-Ficoll). Anti-rat Ig was generatedagainst the anti-CD180 mAb but is not more than 15% of the total IgMproduced and has substantially different kinetics, with Ag-specific IgMrequiring 7 days to peak while total IgM is near maximal by day 3.Class-switched anti-rat Ig was predominately IgG2c and was not producedagainst control mAb. T cell-deficient mice also produced IgM specificfor anti-CD180, but not class-switched Ig of any subclass. Auto-reactiveantibody, as determined by antinuclear antibody immunoflourescence, didnot increase after anti-CD180 injection (data not shown).

Anti-CD180 Injection Expands Splenic B Cells

Three days after injection the spleens of anti-CD180-treated mice wereenlarged nearly 3-fold compared to control mice (data not shown).Absolute splenic mononuclear cell numbers increased approximately2.5-fold from controls (FIG. 12A, B). B cells (CD19⁺) contributed themajority of the change by expanding 7, 9, and 2.5-fold in transitional 1(T1), transitional 2 (T2), and follicular (FO) subsets respectively,while the marginal zone (MZ) B cell subset did not change significantly(FIG. 12A). This selective expansion is not predicted by CD180expression as A) T1 and T2 B cells show equivalent expression with theFO subset but much more accumulation post injection, and B) both CD5⁺ Bcells and a subset of MZ B cells (42%) express CD180 but neitherexpanded following injection (FIG. 12A, C, and data not shown).Furthermore, T cell numbers also expanded significantly (FIG. 12A),despite their lack of CD180 expression, indicating an indirect effect ofanti-CD180 on T cell functions in vivo.

This lymphoid cell expansion after anti-CD180 injection was transient,as cell numbers were lower at day 7 and essentially normal by day 14.The single exception was CD8⁺ T cells, which remained expanded throughday 14. The kinetics of cell expansion paralleled binding of theanti-CD180 antibody, as determined by anti-rat IgG staining ex vivo,which demonstrated maximum binding at day 3, minimal binding at day 7,and undetectable binding on day 14 (data not shown). Expansion of Bcells was still evident in TCR KO mice (FIG. 12B) and showed equivalentkinetics (data not shown), indicating that T cells are not required foreither expansion or contraction B cell populations in vivo followinganti-CD180 injection. However, T cell expansion requires B cells, sinceT cells did not expand in B cell-deficient mice after anti-CD180treatment (FIG. 12B).

Combinations of TLR and CD180 Signals Reduce B Cell Differentiation andEnhance Proliferation

Due to the known interaction between CD180 and TLR4, we compared Igproduction induced by anti-CD180 alone to co-injection with various TLRligands. Combinations of anti-CD180 and LPS did not augment but insteadresulted in decreased or unchanged Ig production (FIG. 13A) resulting inserum concentrations intermediate to anti-CD180 or LPS alone. Thiseffect was also observed with Pam₃CSK₄ (TLR2:1) and CpG (TLR9),indicating a general effect of TLR signals rather than a specificinteraction between CD180 and TLR4.

We also injected anti-CD180 in combination with TLR ligands (LPS or CpG)to determine whether these combinations changed how splenic lymphocytesexpanded. Compared with anti-CD180 injection alone, mice injected withanti-CD180/TLR agonist combinations showed roughly equivalent B cellexpansion (3.5 fold) but had reduced expansion of T cells (FIG. 13B).Despite a lack of MZ B cell expansion with anti-CD180 alone,combinations of CD180 and TLR signals increased splenic MZ B cellpopulations. As splenic lymphocyte analysis conflates proliferation,apoptosis, selection alteration, and tissue homing for a qualitativeresult we are unable to quantitatively assess the effect of CD180/TLRcombination stimulation on B cell proliferation in isolation—leading usto examine the effects of CD180/TLR stimulant combinations in defined invitro experiments.

As CD180 KO B cells have diminished proliferative responses to LPS²², weexamined possible reciprocal dependence of CD180 signals on TLR2 andTLR4. In WT splenocyte cultures the combination of CD180 and TLR4stimulation augmented B cell proliferation compared to either stimulusalone, increasing both the percentage of B cells proliferating and theaverage number of cycles (FIG. 13C). Deficiency of TLR2 and TLR4 had noeffect upon proliferation of B cells in response to anti-CD180, and asexpected there was no LPS response. Similar results were obtained forMyD88 KO B cells (data not shown). Thus, CD180 and TLR4 providedistinct, non-redundant, and mutually reinforcing signals for B cellproliferation.

To identify the intersection of CD180 and TLR4 signaling pathways weassayed B cell proliferation with graded doses of LPS, with or without afixed dose of anti-CD180, in splenocytes from WT, TRIF KO, and MyD88 KOmice (FIG. 13D). Despite the minimal proliferation to LPS alone, theaugmentation of anti-CD180 on LPS-induced proliferation was stillpresent in B cells from TRIF KO mice but not from MyD88 KO mice. WhileMyD88 is not required for CD180 signals to induce B cell proliferation,it is required to mediate the CD180 augmentation of TLR4 signals.

Anti-CD180 Synergizes with Multiple MyD88-Dependent TLR Ligands for BCell Proliferation

Since Ig production decreased after various TLR ligands were co-injectedwith anti-CD180, we examined the effects of TLR ligands on anti-CD180induced proliferation in a quantitative in vitro system designed todetermine the nature and magnitude of signal interactions. B cells wereisolated and both anti-CD180 and TLR agonists were titrated, firstseparately and then together at a constant ratio, to measure interactioneffects between the stimuli. In addition to TLR4 (LPS), the interactionsof CD180 with TLR2:1 (Pam₃CSK₄), TLR2:6 (Pam₂CSK₄), TLR7 (CL097), TLR9(CpG ODN1826), and BCR (anti-IgM F(ab′)₂) were also analyzed (FIG. 14A).The proliferation of B cells to combinations of anti-CD180 mAb and TLRagonists was augmented for all combinations; however, the augmentationwas most pronounced with TLR2 ligands. In contrast, antagonism wasobserved with BCR stimulation.

To extract quantitative information from the titration series ofanti-CD180 and TLR (or BCR) interaction, the three separate titrationcurves were transformed into a single curve (FIG. 14B) by theCombination Index (CI) analysis method²¹. The resulting graph displayssignal interaction over the entire titration range, with CI=1 indicatingno interaction (mere additive effect), CI<1 indicating synergy (greaterthan additive effect), and CI values>1 indicating antagonism. Despitethe previously reported selective relationship between CD180 and TLR4,we demonstrate synergy (CI<1) for all MyD88-dependent TLR agonistcombinations with anti-CD180. Surprisingly, at very low FractionalEffects (relative doses) all combinations other than LPS revealedantagonism. While CD180 is described as a specific regulator of TLR4,our analyses show significantly greater synergy with ligands of TLR2 orTLR7. As these experiments used isolated B cells (>99% pure) theobserved interactions are likely intrinsic to B cells, rather thanindirect contributions from signaling in rare non-B cells.

Anti-CD180 Augments Cytokine Production by TLRs in Isolated B Cells

We next examined cytokine production from isolated B cells treated withanti-CD180 alone or in combinations with LPS or CpG (FIG. 15A). Noproduction of IL-6, IL-10, or TNF-α was observed with anti-CD180 alone;however there was clear augmentation of cytokine production incombination with TLR ligands. Concentrations of IL-6 and IL-10 weresubstantially augmented even at LPS concentrations that alone resultedin no effect. Similar augmentation of cytokine production was seen incombinations with CpG and included strongly increased production ofTNF-α. Isolated DCs similarly did not produce cytokines after CD180stimulation alone (FIG. 15B). Rather than the enhancement with CD180/TLRcombinations observed in isolated B cells, DCs tended to have reducedcytokine production.

Discussion

Collectively, our data indicate that CD180 signals induce an extensiveand rapid burst of polyclonal proliferation and activation in naïve Bcells, and proceeding to IgG production within three days. CD180 hasbeen implicated in induction of IgG3 antibodies since constitutive serumconcentrations of IgG3 in CD180 KO mice are approximately one-tenth thatof WT mice²². Our results broaden this interpretation as anti-CD180 mAbinjection caused very rapid and large increases in serum Igconcentration, with IgM, IgG1, IgG2c, and IgG3 concentrations eachreaching or exceeding 1 mg/ml within 10 days of injection. IgG3concentrations had the largest change with a >50-fold increase overbasal concentrations. While robust, this response was transient asconcentrations of all isotypes had peaked and begun to decline by day14.

Injection of anti-IgD similarly induces polyclonal B cell activation andproduction of high serum IgG1 concentrations^(23, 24), and is theclosest known parallel for the effects of anti-CD180 in vivo. However,there are notable differences between the effects of anti-CD180 andanti-IgD. Anti-IgD induced polyclonal Ig was restricted to IgG1 and IgEisotypes, and required T cell help and IL-4^(25, 26). In contrast,anti-CD180 injection increased serum concentration of all isotypes andsubclasses except for IgG2b and IgA, the two prototypic TGF-β induced Igclasses²⁷—an effect that required neither T cells nor MyD88-dependentsignaling. As B cell class-switch recombination is thought to requireeither T cell help or MyD88-dependent TLR/TACI signals²⁸,anti-CD180-induced Ig production may involve an unrecognized pathway forclass-switch induction. Notably, anti-CD180 treatment is remarkableamong known polyclonal activators by virtue of its profound and rapidinduction of diverse Ig classes and subclasses. Additionally, while Igproduction by anti-IgD required higher order clustering produced byeither multiple mAbs or polyclonal sera^(23, 25), a single anti-CD180mAb induces extensive Ig production, which suggests that only ligationor dimerization is required. While our data do not support the idea ofCD180 signaling via IgD, we cannot rule out the involvement of BCRcomponents²⁹. Despite the significant differences between CD180 and IgDas mediators of polyclonal activation, they still may be classifiedtogether in that both induce potent effects but have no confirmedfunction despite their discovery over 20 years ago.

The anti-CD180-induced Ig is polyclonal and not merely the result of anunexamined Ag-specific response. As rapid production of Ag-specific Igcan occur with either TI-1 or TI-2 antigens, and cellular debris maystimulate B cells for these responses, we examined the effect ofanti-CD180 on NP-conjugated model antigens. It is unlikely that cellulardebris is stimulating Ig production as Ag-specific antibody was reducedfor both responses. Also, antinuclear antibody did not increase withanti-CD180 treatment (data not shown). While Ag-specific responses toindependent but co-administered Ag decreased, Ig specific for theanti-CD180 mAb itself was increased, though not to more than 15% of thetotal IgM produced. As the bulk of the IgM and essentially all of theIgG produced by anti-CD180 treatment is neither memory nor specific forconcomitantly present antigens, it is therefore likely to be polyclonal.

Injection of anti-CD180 mAb resulted in a rapid increase in spleniccellularity; three days after injection T1, T2, and FO B cell subsetsexpanded 7-, 9-, and 2.5-fold, respectively, whereas neither MZ nor CD5⁺B cells expanded. While these lymphocyte expansions conflate survivaland tissue homing effects with proliferation, the magnitude is difficultto explain on the basis of enhanced survival or redistribution alone andmost likely involve a component of proliferation. While T cells do notexpress CD180 or proliferate after anti-CD180 stimulation in vitro,their numbers are significantly increased in the spleen followinganti-CD180 injection, suggesting expansion and/or recruitment of T cellsto the spleen driven by other, directly activated, cells. Regardless ofthe mechanism, the expansion of T cells in the spleen is abrogated in Bcell-deficient μMT mice, indicating that activated B cells and not otherCD180⁺ cells (DC, macrophage) are required for the effect. Increases toboth B and T cell numbers were transient, approaching normal numbers byday seven after injection; only CD8⁺ T cell numbers remained increasedthrough day 14. The function of these persistent CD8⁺ T cells isunknown, as anti-CD180-induced expansion and contraction of B cells wereequivalent in WT and T cell-deficient mice. It is possible that theprevalence of activated B cells is mediating memory T cell reactivationwithout the presence of cognate antigen.

The combined injection of anti-CD180 with LPS, both inducers ofpolyclonal Ig, did not further increase Ig in serum, but insteadresulted in a reduction of Ig levels to concentrations intermediate tothose seen with either stimulus alone. A similar effect was seen withco-injection of anti-CD180 with either TLR9 or TLR2:1 ligands (CpG orPam₃CSK₄). The suppression of anti-CD180 induced Ig by different TLRligands suggests either a restraining effect of non-B cells or anintrinsic negative regulation by TLR signals of CD180 stimulated B celldifferentiation upon the integration of TLR signals. Our data support amodel where combinations of CD180 and TLR signals drive greater B cellproliferation at the expense of differentiation and Ig production.

Our data regarding B cell proliferation to anti-CD180 and LPS are notconsistent with models suggesting CD180 functions by formingheterodimers only with TLR4 and regulating the canonical LPS signal⁴.Unlike LPS, the B cell proliferative response to anti-CD180 does notrequire MyD88, TRIF, or TLR4, and also TLR2 is not required. However,CD180 and TLR signals appear to be integrated through MyD88 because thecombination of anti-CD180 and LPS signals augments B cell proliferationin TRIF-deficient but not MyD88-deficient B cells. Taken together, theseresults indicate that CD180 signals augment, but are independent from,those of TLR4. Given these findings, we hypothesized that otherMyD88-dependent TLRs (e.g. TLR9, TLR7, TLR2:6, and TLR2:1) would alsoenhance B cell proliferation in response to CD180 ligation. Indeed,strong augmentation was evident with anti-CD180 and all TLR ligandstested. This effect may not have been detected in previous studies,which used only single concentrations of ligand combinations; saturationconcentrations may have resulted in an insignificant augmentation unlikesub-maximal doses. As TLR7 and TLR9 are largely endosomal³⁰, and not atthe cell surface where CD180 is found, our data are not consistent witha model of CD180 function involving direct interactions with TLRs toaugment B cell proliferation.

Our analysis allowed the use of the mathematical transformationdescribed by Chou and Talalay²² to quantify synergy over broad doseranges. Synergy is highest between anti-CD180 and the TLR2 ligands,followed by TLR7, then by TLR9, with the least synergy between CD180 andTLR4. The analysis also revealed previously unreported antagonismbetween anti-CD180 and all MyD88-dependent TLR ligands, excluding LPS,at very low doses. Neither of these patterns is predicted by existingmodels of CD180 as a selectively forming heterodimers with TLR4,regardless of whether the interaction is stimulatory or inhibitory.Regardless of whether CD180 acts as a specific TLR4 “decoy” receptor inB cells, as proposed for DCs³, or a required co-receptor for a single Bcell LPS pathway²², the effect should impact both the MyD88 and TRIFsignaling pathways for LPS and no effect would be expected for otherTLRs. Thus, our findings showing that CD180 synergizes with multiple TLRligands in a MyD88-dependent TRIF-independent manner to enhanceproliferation at nearly all dose levels suggest an alternative modelwhere independent CD180 and TLR signals converge in B cells at the levelof MyD88.

While anti-CD180 stimulation of purified B cells induced proliferation,it did not induce cytokine production. However, in combination with LPS,anti-CD180 stimulation increased production of IL-10 and IL-6, but notTNF-α, while anti-CD180 plus CpG increased production of all of thesecytokines. The IL-10 concentrations were high (>1,000 pg/ml), suggestingthat CD180 signals could be involved in development of anti-inflammatoryIL-10 secreting B cells³¹. Due to the complex effects of IL-10, whichcan both suppress inflammation and activate B cells^(32, 33), it ispossible that combined CD180/TLR signaling may minimize TLR-inducedinflammation while promoting select B cell functions. As with B cells,DCs failed to produce cytokines with anti-CD180 stimulation alone,however unlike B cells they did not augment TLR-induced cytokineproduction. A combination of evidence regarding anti-CD180 treatment—thelack of DC responsiveness, the requirement of B cells for splenicexpansion, the production of high serum concentrations of Ig in both WTand T cell-deficient mice, and the proliferation of purified B cells invitro—together suggest that CD180 stimulation is primarily mediated by,and intrinsic to, B cells.

Our study of CD180 is unique in that use of an agonistic antibody allowsus to perform quantitative interaction assays over broad dose ranges andcharacterize acute responses as opposed to genetic deletion that resultsin data that is singular in both dose and kinetics. Taken together, ourresults suggest that CD180 stimulation plays an important role in B cellproliferation, activation, and differentiation, and that these effectsare significantly modulated by integration of MyD88-dependent TLRsignals. While it remains to be determined whether the rapidly inducedclass-switched Ig also involves somatic hypermutation, it appears to bepolyclonal. Finally, because anti-CD180 treatment inducesimmunomodulatory effects (augmenting anti-inflammatory IL-10, bluntingAg-specific responses, and producing polyclonal Ig which may clearapoptotic debris like natural antibody) it has therapeutic potential insystemic autoimmune diseases.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

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We claim:
 1. An isolated anti-CD180 antibody or an antigen bindingfragment thereof, comprising the complete amino acid of SEQ ID NO:2.